Urinalysis device and dry reagent for quantitative urinalysis

ABSTRACT

There is disclosed herein a method of quantitatively determining the concentration of at least one analyte in a sample, the method comprising the steps of either: (i) adding a portion of the sample to a first analyte assay formulation and to an analyte assay reference formulation to generate a first analyte sample and analyte reference sample and determining the concentration of the at least one analyte in the sample and/or (ii) adding a portion of the sample to a second analyte assay formulation and determining the concentration of the at least one analyte in the sample. Also disclosed herein are formulations, kits of parts, systems and computer implemented methods associated with said method.

BACKGROUND

Urinalysis is a source of information about the anatomy and function ofthe kidneys and urinary tract. It provides insights into the status ofsystemic diseases such as diabetes mellitus. The use of test strips, ordipsticks, for urinalysis is widely accepted for health screeningpurposes because it provides a simple protocol and is verycost-effective.

Dipstick urinalysis is convenient, but false-positive and false-negativeresults can occur due to the discrimination of color change when it isperformed by a human eye, such as by a nurse.

Chronic Kidney Disease (CKD) can be diagnosed in its early stages by amicroalbuminuria measurement. The “golden standard” for quantitativemeasurement of microalbuminuria is from a 24-hour urine collection, butthis is very time-consuming and troublesome for patients. Instead, arandom spot urine test is most commonly used to screen microalbuminuria,which requires measuring the urine albumin creatinine ratio (ACR), usingcreatinine to compensate for variations in urine concentration in urinesamples.

ACR is also a useful parameter to measure for use as a prognosis factorfor kidney failure risk. Monitoring the ACR value during treatment isalso useful, and a point-of-care device is required to improve patients'Quality of Life (QOL).

Currently, most of the point-of-care devices available in the market forurinalysis are semi-quantitative and use a test strip and strip reader.A quantitative point-of-care device is desired for home or clinical use.

For example, quantitative urinalysis results can be obtained by using anautomatic urine analyzer at the hospital and in a clinical laboratory. Aurine analyzer is generally a desk-top machine or part of a larger pieceof equipment, such as a fully automated blood serum/urine analyzer.Therefore, the accessibility of these quantitative urinalysis units iscentralized and people who live in rural areas have limited access toproper diagnostic tests.

Urine samples have a large pH variation, and the measurement results arealso affected by the temperature of the surroundings (i.e. the testingplace). Current quantitative assay methods require sample dilution toremove the interference caused by urine. In addition, colorimetric wetreagents for urinalysis are required to be kept in refrigerator, whichis not suitable for home or clinical use.

As indicated above, to minimise the effect of pH or interference incurrent quantitative systems, a urine sample is diluted with a volume ofbuffer in addition to a strong acid or a strong alkali. To minimise theeffect of temperature in the testing room, a cooling system isintroduced into the device used for analysis, but this cooling systemmakes the device bulky. The size of the resulting device makes itdifficult to transport and renders it unusable in anything other than aclinical laboratory setting.

In addition, the reagents used in urinalysis may suffer from bubbleformation, either occurring during the reaction, or from the addition ofthe sample to the reagent. This can result in the loss of accuracy dueto light scattering.

For current quantitative devices, an internal reference table isnormally prepared under a range of pH and/or temperature conditions, soas to normalize the data, but it is troublesome to prepare the referencetable and there is a limit on how far the normalisation can be taken,depending on the type of interference. Given this, the result is notaccurate for all patients.

As will be appreciated, these problems arise, at least in part, becauseof the reagents used in the currently available tests.

Given the above, there remains a need for more accurate urinalysisequipment that is portable and which is cost-effective. In addition,there remains a need for analytical reagents that are capable of beingtailored for use with a simplified device and which help to increase theaccuracy of the result.

SUMMARY OF INVENTION

Aspects and embodiments of the invention are set out in claims 1 to 63,and are considered in more detail below.

In a first aspect of the invention, there is provided a method ofquantitatively determining the concentration of at least one analyte ina sample, the method comprising the steps of:

-   -   i) (a) adding a portion of the sample to a first analyte assay        formulation comprising a first analyte complexing reagent to        generate a first analyte sample;        -   (b) adding a portion of the sample to an analyte assay            reference formulation that is identical to the first analyte            assay formulation, except that it further comprises a            reagent that denatures the analyte or blocks the formation            of a complex between the analyte and the first analyte            complexing reagent, to generate an analyte reference sample;            and        -   (c) determining the concentration of the at least one            analyte in the sample by measuring the absorbance spectra of            the first analyte sample and analyte reference sample,            calculating the difference between the absorbance spectra of            the first analyte sample and the analyte reference sample            and comparing the difference spectrum obtained to a first            pre-determined calibration curve of analyte concentration;    -   and/or    -   ii) (a) adding a portion of the sample to a second analyte assay        formulation comprising a second analyte complexing reagent to        generate a second analyte sample; and        -   (b) determining the concentration of the at least one            analyte in the sample by measuring the absorbance spectra of            the analyte in the second analyte sample over a period of            time, calculating the rate of the absorbance change of the            second analyte sample over said period of time and comparing            the rate obtained to a second pre-determined calibration            curve of analyte concentration.

In an embodiment of the invention, the at least one analyte is albuminand the method comprises the steps of:

-   -   adding a portion of the sample to each of an:    -   A) albumin assay sample formulation comprising an albumin        complexing reagent to generate an albumin sample; and    -   B) albumin assay reference formulation that is identical to the        albumin assay formulation except that it further comprises an        anionic surfactant to generate an albumin reference sample, and    -   determining the concentration of albumin in the sample by        measuring the absorbance spectra of the albumin sample and the        albumin reference sample, calculating the difference between the        absorbance spectra of the albumin sample and the albumin        reference sample and comparing the difference spectrum obtained        to a pre-determined calibration curve of albumin concentration.

In an alternative embodiment of the invention, the at least one analyteis creatinine and the method comprises the steps of adding a portion ofthe sample to a creatinine assay sample formulation comprising acreatinine complexing reagent to generate a creatinine sample anddetermining the concentration of creatinine in the sample by measuringthe change of the absorbance spectra of the creatinine sample over aperiod of time, calculating the rate of the absorbance change of thecreatinine sample over said period of time and comparing the rateobtained to a pre-determined calibration curve of creatinineconcentration.

In a further embodiment of the invention, the at least one analyte isalbumin and creatinine, the method comprising the steps of:

-   -   a) determining the concentration of creatinine in the sample in        accordance with the methods defined above; and    -   b) determining the concentration of albumin in the sample in        accordance with the methods defined above; and    -   further comprising the step of determining the        albumin/creatinine ratio of the sample.

In certain embodiments of the invention, one or more of the formulationsmay be lyophilised. For example, all of the formulations arelyophilised.

In certain embodiments of the invention, the sample is a urine sample.

In further embodiments of the invention, the step of determining theconcentration of albumin requires measuring the absorbance spectra:

-   -   at 625 nm of the albumin sample and albumin reference sample;        and/or    -   at time of about 5 minutes from the addition of the sample to        the desired analyte formulation.

In yet further embodiments, the step of determining the concentration ofcreatinine requires measuring the rate of absorbance change at 525 nm ofthe creatinine sample.

In yet further embodiments, the step of determining the concentration ofcreatinine requires measuring the rate of absorbance change at 525 nm ofthe creatinine sample involves measuring the absorbance during a periodof from about 10 seconds up to about 10 minutes. For example, suchkinetic measurement starts at 30 seconds and ends at 10 minutes from theaddition of the sample to the second assay formulation or the creatinineassay formulation (i.e. the kinetic measurement starts at 1 minute andends at a time between 5 and 10 minutes from the addition of the sampleto the second assay formulation or the creatinine assay formulation).

In yet further embodiments of the invention, the method may determinethe creatinine concentration and the albumin concentrationsimultaneously.

In further embodiments of the invention, the creatinine sample assayformulation is as defined in the second aspect of the invention.

In yet still further embodiments of the invention, the albumin assayformulation is as defined in the third aspect of the invention.

In further embodiments of the invention, the pre-determined calibrationcurve of albumin concentration is obtained by a computer-implementedmethod of generating a calibration curve for use in quantitativelydetermining the concentration of albumin in a urine sample, the methodbeing defined in the sixth aspect of the invention.

In a second aspect of the invention, there is provided a formulation foruse in the analysis of the creatinine concentration of a samplecomprising:

-   -   a strong base in a concentration of from about 40 to about 80        g/L;    -   a buffer in a concentration of from about 50 to about 250 g/L;    -   at least one surfactant in a concentration of from about 0.1 to        about 20 g/L;    -   and water.

In an embodiment of the invention, the formulation further comprises acompound that reacts with creatinine to generate a creatinine complex.For example the compound that reacts with creatinine to generate acreatinine complex is picric acid or, more particularly, dinitrobenzoicacid.

In certain embodiments, the concentration of the compound that reactswith creatinine to generate a creatinine complex is from about 1 toabout 5 g/L.

In further embodiments of the invention, the at least one surfactantcomprises an anionic surfactant in a concentration from about 0.1 toabout 10 g/L and a cationic surfactant in a concentration from about 0.1to about 10 g/L.

In certain embodiments, the ratio of cationic surfactant: anionicsurfactant is from 1:2 to 1:10 (e.g. from 1:3 to 1:7, such as 1:5).

In certain embodiments of the invention, in relation to the formulationfor use in the analysis of the creatinine concentration of a sample:

-   -   (a) when the at least one surfactant comprises a cationic        surfactant, it is selected from one or more of the group        consisting of cetylpyridinium chloride, benzalkonium chloride,        benzethonium chloride, dimethyldioctadecyl-ammonium chloride,        cetrimonium bromide, dioctadecyldimethylammonium bromide, and        hexadecyltrimethylammonium bromide; and/or    -   (b) when the at least one surfactant comprises an anionic        surfactant, it is selected from one or more of the group        consisting of sodium dodecyl sulfate, polystyrene sulfonates,        linear alkylbenzene sulfonate, secondary alcohol sulfonate,        alcohol olefin sulfonate, and alcohol sulfate; and/or    -   (c) the buffer is selected from one or more of the group        consisting of K₂HPO₄, Na₂HPO₄, and borate; and/or    -   (d) the strong base is NaOH and/or KOH.

In certain embodiments of the invention, the formulation for use in theanalysis of the creatinine concentration of a sample is lyophilised.

In yet further embodiments of the invention, the formulation for use inthe analysis of the creatinine concentration of a sample furthercomprises a bulking agent in an amount of from about 1 to about 40weight % of the formulation, optionally wherein the bulking agent isselected from one or more of the group consisting of sugar-mannitol,lactose, and trehalose.

In still further embodiments of the invention, the formulation for usein the analysis of the creatinine concentration of a sample, theconcentration ratio of the buffer:strong base in the formulation is from0.5:1 to 2.5:1, such as from 1:1 to 1.5:5, such as 1.25:1.

In a third aspect of the invention, there is provided a formulation foruse in the analysis of albumin concentration of a sample comprising:

-   -   a compound that reacts with albumin to generate an albumin        complex in a concentration of from about 0.1 to about 1.5 g/L;    -   a strong base in a concentration of from about 10 to about 50        g/L;    -   a buffer in a concentration of from about 50 to about 250 g/L;    -   a first non-ionic surfactant in a concentration of from about 1        to about 20 g/L;    -   a preservative in a concentration of from about 0.1 to about 3        g/L; and    -   water.

In embodiments of the invention, the formulation further comprises ananionic surfactant. In certain embodiments, the anionic surfactant ispresent in from about 0.1 to 5 weight % of the formulation. For example,the anionic surfactant is selected from one or more of the groupconsisting of sodium dodecyl sulfate, polystyrene sulfonates, linearalkylbenzene sulfonate, secondary alcohol sulfonate, alcohol olefinsulfonate, and alcohol sulfate.

In certain embodiments of the invention, the compound that reacts withalbumin to generate an albumin complex is bromocresol green.

In certain embodiments of the invention, in relation to the formulationfor use in the analysis of the albumin concentration of a sample:

-   -   (a) the buffer is selected from one or more of the group        consisting of N-(1-acetamido)-2-aminoethanesulfonic acid, sodium        acetate, N-(2-acetamido)-iminodiacetic acid,        2-amino-2-methyl-1-propanol, 2-amino-2-methyl-1,3-propanediol,        N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxpropanesulfonic        acid, N,N-bis-(2-hydroxyethyl)-2-aminoethane-sulfonic acid,        sodium hydrogen carbonate, N,N-bis-(2-hydroxyethyl)-glycine,        bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane,        1,3-bis-[tris-(hydroxymethyl)-methylamino]-propane,        3-cyclohexylamino)-1-propanesulfonic acid,        3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid,        2-(N-cyclohexylamine)-ethanesulfonic acid, tri-sodium citrate,        N,N-bis-(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid,        4-(2-hydroxyethyl)pipera-zine-1-ethanesulfonic acid,        4-(2-hydroxyethyl)-piperazine-1-propanesulfonic acid,        4-(2-hydroxyethyl)-piperazine-1(2-hydroxy)-propane-sulfonic        acid, 2-morpholinoethanesulfonic acid,        3-morpholinopropanesulfonic acid,        3-morpholino-2-hydroxypropanesulfonic acid,        piperazine-1,4-bis-(2-ethanesulfonic acid),        piperazine-1,4-bis-(2-hydroxy-propanesulfonicacid),        N-[tris-(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid,        N-[tris-(hydroxymethyl)-methyl]-3-amino-2-hydroxypropanesulfonic        acid, N-[tris-(hydroxymethyl)-methyl]-2-aminoethanesulfonic        acid, N-[tris-(hydroxymethyl)-methyl]-glycine,        tris-(hydroxy-methyl)-aminomethane, and succinic acid; and/or    -   (b) the first non-ionic surfactant is selected from one or more        of the group consisting of brij, poly(propylene glycol),        polyethylene glycol hexadecyl ether, triton X-100, tween, Zonyl™        FSN fluorosurfactant, ALKANOL™ 6112 surfactant, polyethylene        glycol, optionally wherein the first non-ionic surfactant is        brij; and/or    -   (c) the preservative is selected from one or more of the group        consisting of sugar, sorbic acid, benzoic acid, calcium        propionate, sodium nitrite, sodium azide, sulfites, disodium        EDTA and antioxidants; and/or    -   (d) the strong base is NaOH and/or KOH.

In further embodiments of the invention, the formulation for use in theanalysis of the albumin concentration (and the reference sample) may belyophilised.

In certain embodiments of the invention, the formulation may furthercomprise a second non-ionic surfactant in an amount of from about 10 toabout 200 g/L of the formulation. For example, the second non-ionicsurfactant may be selected from one or more of the group consisting ofbrij, poly(propylene glycol), polyethylene glycol hexadecyl ether,triton X-100, tween, Zonyl™ FSN fluorosurfactant, ALKANOL™ 6112surfactant, polyethylene glycol, provided that the first and secondnon-ionic surfactants are different to each other, optionally whereinthe second non-ionic surfactant is polyethylene glycol.

In a fourth aspect of the invention, there is provided a method ofpreparing the lyophilised formulation for use in the analysis ofcreatinine concentration of a sample, as described in respect of thesecond aspect of the invention or the lyophilised formulation for use inthe analysis of albumin concentration of a sample as described in thethird aspect of the invention, comprising the steps of:

-   -   (a) mixing the prescribed chemical components together,        filtering the resultant mixture, dispensing said mixture into a        container and inserting the container into a freeze drying        apparatus;    -   (b) freezing the sample at a temperature of from about −20° C.        to about −80° C. for a period of time ranging from about 0.5        hours to 5 hours;    -   (c) annealing the sample at a temperature of from about −10° C.        to about −30° C. for a period of time ranging from about 1 hour        to 5 hours;    -   (d) re-freezing the sample at a temperature of from about        −20° C. to about −80° C. for a period of time ranging from about        0.5 hours to 5 hours;    -   (e) conducting a first drying cycle at a temperature of from        about −10° C. to about −30° C. for a period of time ranging from        about 5 hours to 50 hours; and    -   (f) conducting a second drying cycle at a temperature of from        about 0° C. to about 60° C. for a period of time ranging from        about 1 hour to 20 hours.

In certain embodiments of the invention, the container is selected fromthe group consisting of an eppendorf tube, a 96-well plate, a commercialcuvette or a cuvette adapted for use with a device comprising a contactimage sensor module.

In further embodiments of the invention, the method may further comprisestoring the container holding the lyophilised formulation away fromlight, oxygen and moisture.

In a fifth aspect of the invention, there is provided a kit of partscomprising:

-   -   (a) a creatinine assay formulation according to the formulation        of the second aspect of the invention; and/or    -   (b) an albumin assay formulation and an albumin reference        formulation according to the formulation of the third aspect of        the invention.

In certain embodiments of the invention, in the kit of parts, eachlyophilised formulation may be individually contained in an eppendorftube, a cuvette, a cuvette adapted for use with a device comprising acontact image sensor module, or separate wells in a 96-well plate.

In certain embodiments, the formulations are stored away from light,oxygen and moisture.

In a sixth aspect of the invention, there is provided acomputer-implemented method of generating a calibration curve for use inquantitatively determining the concentration of albumin in a urinesample, the method comprising the steps of:

-   -   obtaining, first absorbance data representing albumin-free        sample absorbances and albumin-free reference absorbances for a        plurality of albumin-free urine samples; wherein the        albumin-free sample absorbances comprise absorbances for        respective first portions of the albumin-free urine samples in        the presence of an albumin-complexing reagent; and the        albumin-free reference absorbances comprise absorbances for        respective second portions of the albumin-free urine samples in        the presence of the albumin-complexing reagent, and additionally        in the presence of an albumin-denaturing reagent or a reagent        that blocks the formation of a complex between albumin and the        albumin-complexing reagent;    -   computationally fitting a functional relationship between the        albumin-free sample absorbances and the albumin-free reference        absorbances to obtain adjustment parameters;    -   obtaining second absorbance data representing sample absorbances        and reference absorbances for a plurality of urine samples        having known concentrations of albumin;

wherein the sample absorbances comprise absorbances for respective firstportions of the urine samples in the presence of the albumin-complexingreagent; and the reference absorbances comprise absorbances forrespective second portions of the urine samples in the presence of thealbumin-complexing reagent, and additionally in the presence of thealbumin-denaturing reagent or the reagent that blocks the formation of acomplex between albumin and the albumin-complexing reagent;

-   -   adjusting the reference absorbances using the adjustment        parameters, to thereby obtain adjusted reference absorbances;        and    -   computationally fitting a functional relationship between the        sample absorbances, the adjusted reference absorbances and the        known concentrations to obtain parameters of the calibration        curve.

In an embodiment of the invention, the functional relationship betweenthe albumin-free sample absorbances and the albumin-free referenceabsorbances is of the form A_(S) ⁰=a*A_(R) ⁰+b, where A_(S) ⁰ are thealbumin-free sample absorbances, A_(R) ⁰ are the albumin-free referenceabsorbances, and a and b are the adjustment parameters.

In a further embodiment of the invention, the adjusted referenceabsorbances are calculated according to a*A_(R) ^(*)+b, where A_(R) ^(*)are the reference absorbances.

In a still further embodiment of the invention, the functionalrelationship between the sample absorbances, the adjusted referenceabsorbances and the known concentrations is of the form A_(S)^(*)−(a*A_(R) ^(*)+b)=A*C_(Alb) ^(*), where A_(S) ^(*) are the sampleabsorbances, C_(Alb) ^(*) are the known concentrations, and A is aparameter of the calibration curve.

DESCRIPTION OF THE ATTACHED DRAWINGS

FIG. 1: Sensing principle of albumin

FIG. 2: Sensing principle of creatinine

FIG. 3: Dry reagent preparation

FIG. 4: Cuvette cartridge and cuvette holder

FIG. 5: Sample introduction device

FIG. 6: Sample introduction structure

FIG. 7: Cuvette holder and optical module

FIG. 8: Assay method

FIG. 9: BCG dry-reagent: Compensation of pH effect

FIG. 10: BCG dry-reagent: Compensation of temperature effect

FIG. 11: Calibration curve of BCG dry-reagent

FIG. 12: Calibration curve of BCG dry-reagent with real urine samplebefore data processing

FIG. 13: Graph of A_(S) versus A_(R)

FIG. 14: Calibration curve of BCG dry-reagent with real urine sampleafter data processing

FIG. 15: Calibration curve of DNBA dry-reagent

DESCRIPTION OF INVENTION

It is desired to provide a simple and compact point-of-care urinalysissystem using wet, or more particularly, dry reagents. In particular, thereagents may be suitable for use with a point-of-care analysis systemthat can simultaneously measure a number of analytical samples.

For example, such a system may comprise the use of a contact imagesensor (CIS) module having a light emitting diode (LED) to emit light,the wavelength of the light is preconfigured to match the absorptionwavelength of the reaction product of a urinalysis reagent and targetanalyte, an optical cuvette having the reflective surface at the farside of the cuvette from the CIS module, a freeze-dried urinalysisreagent being disposed in the optical cuvette, a urine distributionstructure having reservoir to distribute sample urine into individualoptical cuvette and having solution stopping structure to control thevolume of distributed solution separately. Chronic Kidney Disease (CKD)can be diagnosed in its early stages by measuring microalbuminuria. Suchtests can be performed by determining urine microalbuminuria andcreatinine concentrations and their corresponding albumin/creatinineratio (ACR).

While the tests used herein may be used to analyse a urine sample, thetests are also suitable for analysing samples from other sources (e.g.saliva, blood etc). As such, the term “urine microalbuminuria” may bereplaced throughout the specification by the term “albumin”.

In order to detect ACR, two different reagents are used for urinemicroalbuminuria and creatinine measurements, respectively. Bromcresolgreen (BCG) is used for urine microalbuminuria detection, while3,5-dinitrobenzoic acid (DNBA) is used for urine creatinine detection.

Microalbuminuria present in a urine sample reacts with BCG to form acolored complex (FIG. 1). The intensity of the color, measured at awavelength of 625 nm, is directly proportional to the microalbuminuriaconcentration in the urine sample.

For the microalbuminuria assay, two types of wet or, more particularly,dry reagents are prepared for sampling and referencing. The mixtures ofthe dry reagents (sampling and referencing) and the urine sample aremeasured concurrently by colorimetric assay. The sampling reagent isused to measure the intensity of the color obtained by the BCG-albumincomplex. The referencing reagent is used to reduce or remove pH andtemperature effects and interference by applying a protein denaturingmethod to the urine sample. That is, the microalbuminuria assay appliesa protein denaturing method, where the dry reagent for referencingcontains a surfactant to denature albumin, so that denatured albumindoesn't react with the BCG present in the referencing reagent.

Creatinine present in a urine sample reacts with DNBA in alkaline mediumto form a purple-red complex (FIG. 2). The rate of complex formation atwavelength of 525 nm is directly proportional to the concentration ofcreatinine in the urine sample. Alternatively, DNBA may be replaced bypicric acid.

The creatinine assay only uses a sampling reagent for measuringcreatinine. The creatinine sampling reagent applies a buffer additionmethod to minimise the interference from urine pH, while the temperatureeffect is compensated by a temperature factor in the calibration curveand the urine background effect is eliminated by measuring the rate ofcomplex formation. Current, devices are tailored so as to operate withinthe reaction parameter of conventional formulations. This may result ina more complicated automated device, as differing sensors may need to beused to analyse the reaction products of the different reagents used todetect analytes in urine (e.g. colourimetric analysis).

As described in more detail below, it is important to control thekinetics of the interaction between DNBA and creatinine. If the reactionbetween DNBA and creatinine is too fast or too slow for the device beingused, then the device will not be able to provide an accurate analysis.In addition, as the mixture of DNBA dry-reagent and urine sample has atendency to form bubbles, the analysis may not be accurate due to lightscattering. The formulations disclosed herein has been formulated inorder to overcome such problems associated with the reagent and theanalysis of the sample.

Dry Reagent for Creatinine Assay

A buffer addition method is applied to the DNBA dry-reagent to removethe effect of urine pH. DNBA reacts with creatinine under alkalineconditions, and a purple-colored complex is generated. The samplingreagent contains DNBA, a strong base, a buffer, a bulking agent, ananionic surfactant and a cationic surfactant. The specific chemicalsused can be chosen from the list below (Table A), and the concentrationof each chemical can be within the specified range.

TABLE A Range of Concentration Chemical Category Creatinine-Samplingreagent Strong base 40-80 g/L Buffer 50-250 g/L Bulking agent 1-40 wt %(10-400 g/L) Anionic surfactant 0.1-10 g/L Cationic surfactant 0.1-10g/L DNBA 1-5 g/L

Chemical List for Creatinine Assay

-   -   1) Strong base; NaOH, KOH.    -   2) Buffer; K₂HPO₄, Na₂HPO₄, Borate.    -   3) Bulking agent; Sugar (e.g. mannitol, lactose, trehalose).    -   4) Anionic surfactant; especially anionic sulphated/sulphonated        surfactant; Sodium dodecyl sulfate (SDS), Polystyrene sulfonates        (PSS), linear alkylbenzene sulfonate (LAS), secondary alcohol        sulfonate, alcohol olefin sulfonate, alcohol sulfate.    -   5) Cationic surfactant; Cetylpyridinium chloride (CPC),        Benzalkonium chloride (BAC), Benzethonium chloride (BZT),        Dimethyldioctadecyl-ammonium chloride, Cetrimonium bromide,        Dioctadecyldimethyl-ammonium bromide (DODAB),        Hexadecyltrimethylammonium bromide (CTAB).

Without wishing to be bound by theory, it is suspected that the additionof a buffer to the strong base results in the kinetic modulation of thechemical reaction between DNBA or picric acid and creatinine. As will beappreciated, the ratio of buffer and sodium hydroxide may be selected tosuit the analytical device. This enables the device to produce a moreaccurate reading. For example, the concentration ratio (in Molar) of thebuffer:strong base may be from 0.5:1 to 3.2:1, such as from 0.8:1 to2.5:1 (e.g. from 1.5:1 to 2.5:1), such as from 1:1 to 1.5:1 (e.g.1.25:1). For example, when used with the device described in FIGS. 4 to7 of the current application, a buffer:strong base ratio of less than0.5:1 may result in the complex formation becoming too quick toaccurately measure the rate of its formation.

In addition, without wishing to be bound by theory; it is believed thatthe addition of surfactants helps to reduce the formation of bubbles onthe optical cuvette wall in the reaction mixture, thereby reducingscattering and increasing accuracy. In particular, it has been foundthat the use of a cationic surfactant that includes a quarternaryammonium moiety is useful in controlling the formation of bubbles whenused in combination with an anionic surfactant as a solvation assistant.This is because the quarternary ammonium surfactant is not able todissolve in sufficient quantity in the buffered solution without asolvation aid (e.g. an anionic surfactant), while the anionic surfactantdoes not affect bubble formation when used alone. Therefore, in order tocontrol bubble formation, it is useful to use a specific ratio ofanionic and cationic surfactants in the creatinine assay reagent. Theratio of the cationic surfactant: anionic surfactant may be from 1:2 to1:10, such as from 1:3 to 1:7 or, more particularly, 1:5.

Dry Reagent for Microalbuminuria Assay

A protein denaturing method is applied for the BCG dry-reagent to removethe interference from the urine components, such as urine pH, sampletemperature and urine background. Albumin reacts with BCG and the colorchanges, but denatured albumin does not react with BCG and the colorremains the same. The 1^(st) reagent is for sampling and the 2^(nd)reagent is for referencing. Both reagents contain the sameconcentrations of BCG, Strong base, buffer, non-ionic surfactant andpreservative. However, only the 2^(nd) reagent contains an anionicsurfactant to denature albumin in the reference sample. The chemicalscan be chosen from the list provided below (Table B), and theconcentrations can be within the specified range.

TABLE B Range of Concentration Albumin- Albumin- Chemical CategorySampling reagent Referencing reagent BCG 0.1-1.5 g/L 0.1-1.5 g/L Strongbase 10-50 g/L 10-50 g/L Buffer 50-250 g/L 50-250 g/L Non-ionicsurfactant 2 1-20 wt % 1-20 wt % (10-200 g/L) (10-200 g/L) Non-ionicsurfactant 1 1-20 g/L 1-20 g/L Preservative 0.1-3 g/L 0.1-3 g/L Anionicsurfactant 0 w % 0.1-5 w % (1-50 g/L)

Chemical List for Albumin Assay

-   -   1) Strong base; NaOH, KOH.    -   2) Buffer; ACES (N-(1-Acetamido)-2-aminoethanesulfonic acid),        Acetate (Sodium acetate), ADA (N-(2-Acetamido)-iminodiacetic        acid), AMP (2-Amino-2-methyl-1-propanol), AMPD        (2-Amino-2-methyl-1,3-propanediol), AMPSO        (N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic        acid), BES (N,N-Bis-(2-hydroxyethyl)-2-aminoethane-sulfonic        acid), Bicarbonate (Sodium hydrogen carbonate), Bicine        (N,N-Bis-(2-hydroxyethyl)-glycine), Bis-Tris        (Bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane),        Bis-Tris-Propane        (1,3-Bis-[tris-(hydroxymethyl)-methylamino]-propane), CAPS        (3-Cyclohexylamino)-1-propanesulfonic acid), CAPSO        (3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CHES        (2-(N-Cyclohexylamine)-ethanesulfonic acid), Citrate (tri-Sodium        citrate), DIPSO        (N,N-Bis-(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic        acid), HEPES (4-(2-Hydroxyethyl)-pipera-zine-1-ethanesulfonic        acid), HEPPS (4-(2-Hydroxyethyl)-piperazine-1-propanesulfonic        acid), HEPPSO        (4-(2-Hydroxyethyl)-piperazine-1(2-hydroxy)-propane-sulfonic        acid), MES (2-Morpholinoethanesulfonic acid), MOPS        (3-Morpholinopropanesulfonic acid), MOPSO        (3-Morpholino-2-hydroxypropanesulfonic acid), PIPES        (Piperazine-1,4-bis-(2-ethanesulfonic acid)), POPSO        (Piperazine-1,4-bis-(2-hydroxy-propanesulfonicacid)), TAPS        (N-[Tris-(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid),        TAPSO        (N-[Tris-(hydroxymethyl)-methyl]-3-amino-2-hydroxypropanesulfonic        acid), TES        (N-[Tris-(hydroxymethyl)-methyl]-2-aminoethanesulfonic acid),        Tricine (N-[Tris-(hydroxymethyl)-methyl]-glycine), Tris        (Tris-(hydroxy-methyl)-aminomethane), Succinic Acid.    -   3) Non-ionic surfactant; Poly(propylene glycol) (PPG),        Polyethylene glycol hexadecyl ether (Brij), Triton X-100, Tween,        Zonyl® FSN fluorosurfactant, ALKANOL® 6112 surfactant,        Polyethylene Glycol (PEG).    -   4) Preservative; Sugar, sorbic acid, benzoic acid, calcium        propionate, sodium nitrite, sodium azide, sulfites, disodium        EDTA, Antioxidants.    -   5) Anionic surfactant; Sodium dodecyl sulfate (SDS), Polystyrene        sulfonates (PSS), linear alkylbenzene sulfonate, (LAS),        secondary alcohol sulfonate, alcohol olefin sulfonate, alcohol        sulfate.

Method of Preparing Dry Reagent (FIG. 3)

The prescribed chemical components are mixed and filtered through a 0.45μm cellulose acetate filter disk. The filtered mixture is dispensed intoan appropriate sample well by pipette or dispenser. The sample well canbe an eppendorf tube, a 96 well plate, a commercial cuvette, or anyother equivalent reagent holder. For example, the sample well may be acuvette cartridge (102) as depicted in FIG. 4. The sample well isinserted into a freeze-dryer and freeze-dried under a specific profile,which consists of freezing, annealing, freezing, primary drying andsecondary drying processed. The freeze-drying process can be performedunder moisture-less conditions to avoid contact with moisture.

The use of other reagent holders may require optimization of the primarydrying duration (e.g. extension of duration).

An example of the freeze-drying profile and the range of parameters isprovided in the table below. These conditions may be particularly suitedto the cuvette cartridge depicted in FIG. 4.

Freeze Drying Duration Vacuum Process Temp [° C.] [hr] [mTorr] Freezing−20 to −80 0.5 to 5 N/A Annealing −10 to −30   1 to 5 N/A Freezing −20to −80 0.5 to 5 N/A Primary drying −10 to −30   5 to 50 50-300 Secondarydrying  0 to 60   1 to 20 50-300

Packaging

The dried reagent should be stored in the dark and in dry conditions toprevent light, moisture and oxygen exposure. The dried reagent can bepacked into moisture barrier bags and vacuum sealed in a glove box (i.e.under an inert atmosphere). If necessary, a desiccant or an antioxidantcan be inserted into the package.

Cuvette Cartridge, Introduction Device and Assay Method

A cuvette cartridge system and sample introduction device can beutilized with the dry reagent (e.g. as depicted in FIGS. 4 to 7). Thewidth of the cartridge may be calculated based on the path length ofeach analyte. Volume can be the same in each cuvette cartridge (102)when the assay is for multiple analytes. For example, the volume can be0.5 mL to 5 mL, preferably 1 mL. For example, if the volume is 1 mL,then the cuvette width can be 5 mm for the Albumin assay, and 2 mm forthe creatinine assay. The cuvette cartridge (102) can be made oftransparent material, such as PMMA (Poly-methyl methacrylate, PC(Polycarbonate), COC (Cyclic Olefin Copolymer). To block stray light,the color may be black.

Liquid reagent is dispensed into a cuvette cartridge (102) by dispenser(e.g. a Musashi Dispenser), and freeze-dried by a Freeze-dryer (VirTisAdVantage Plus Freeze-dryer). For example, using the method of preparingand storing the dry reagent used above. It will be appreciated that thereagent may be used in “wet” form, that is, without freeze-drying. Thecuvette cartridge (102) is transferred into a cuvette holder (100; FIG.4).

As shown in FIG. 4, the cuvette holder (100) can hold 4 cuvettecartridges (102). For example 4 cuvettes can be arranged in 2 linesseparated by a light blocking structure (101). 2 cuvette cartridges(102) can be used for one analyte, and the other 2 cuvette cartridgescan be used for the other analyte. As shown in FIG. 7, two opticalmodules (700) can be arranged to each side of the cuvette holder (100).It will be appreciated that the cuvette holder (100) also serves tomaintain the distance between the cuvette (102) and the light module(700), so that a certain path length is maintained during samplemeasurement.

Urine is collected from a subject into a collection cup, sucked up intoa syringe and introduced into a sample well (501; FIG. 5) through asample introduction port (500) in a sample well cover (502). Urine isdispensed into each cuvette (102) through a sample introduction hole(600; FIG. 6). Urine and dry reagent are mixed in the cuvette cartridgeby shaking, magnetic stirring, vibration, sonication, or in any otherequivalent way. Optical measurement is performed using one or moreoptical modules (700; FIG. 7). As shown in FIG. 8, the absorbance valueis measured for each cuvette, and, where necessary, the differencebetween the sample and reference is calculated. By utilizing acalibration curve which has been prepared beforehand, the analyteconcentration can be calculated, and the albumin to creatinine ratio canbe calculated thereafter.

FIGS. 9-11 show a calibration curve for the microalbuminuria assay basedupon synthetic standards (e.g. a synthetic urine matrix). These albuminstandards are prepared with artificial or pooled urine and a certainconcentration of albumin. FIGS. 9A and 10A use the sampling reagent,FIGS. 9B and 10B use the referencing reagent, FIGS. 9C, 10C and 11 showthe calibration curve, which results from the difference between thesampling reagent and referencing reagent when contacted with, thestandards. Table 1 shows the results obtained before normalization ofthe results in FIG. 9 for pH variations, while Table 2 shows the resultsobtained after normalization using equation (1) below. Table 3 shows theresults before normalization found in FIG. 10, while. Table 4 providesthe data normalized to compensate for temperature effects.

TABLE 1 Estimated [Albumin] in mg/L [Albumin] pH 5.08 pH 6.65 pH 8.07 0−55.81 3.93 54.19 20 −39.66 20.00 77.35 50 −8.89 49.91 104.96 100 32.82100.77 151.79 200 139.15 200.43 241.71

TABLE 2 Estimated [Albumin] in mg/L [Albumin] pH 5.08 pH 6.65 pH 8.07 00.51 −2.73 −0.32 20 24.12 19.21 23.94 50 49.40 50.14 55.97 100 94.49100.51 101.62 200 196.62 198.94 201.81

TABLE 3 [Alb] 25° C. 28° C. 31° C. 34° C. 0 −2.20 6.83 17.56 36.10 2018.78 27.32 37.32 55.61 50 48.78 58.29 67.80 85.85 100 103.90 110.98120.00 137.07 200 198.29 204.63 213.17 229.51

TABLE 4 [Alb] 25° C. 28° C. 31° C. 34° C. 0 −1.05 −1.05 1.77 5.36 2019.46 17.41 19.72 19.46 50 46.64 47.41 49.72 53.31 100 106.38 102.54104.33 106.64 200 198.18 195.36 196.13 197.15

A _(S) −A _(R) =A*C _(Alb) B  (1)

Where A_(S) refers to the sampling reagent, A_(R) refers to thereferencing reagent, C_(Alb) refers to the concentration of albumin andA and B are constants. While the resulting calibration curve was foundto be accurate for subjects with medium to high levels of albumin (e.g.30-300 mg/L), it was found that the results obtained for low levels ofalbumin (e.g. 0-30 mg/L) could vary significantly. These results aredepicted in FIG. 12 and in Table 5 (i.e. the unspiked urine samplesshould have an average concentration of around 0 mg/L). It is believedthat this inaccuracy is caused by a urine matrix side-effect, whichresults in greater variation at lower concentrations.

TABLE 5 Quantified [Albumin] Spiked Vol. # Un-spiked 100 mg/L 1 14 102 218 116 3 8 104 4 5 101 5 3 99 6 6 104 7 14 107 8 15 105 9 8 105 10  −1101 Average 9 104 Stdev 6.20 4.77

In order to improve the accuracy of the assay at low albuminconcentrations in real human samples, it was necessary to conductfurther processing, as described below.

Original calibration curve:

A _(S) −A _(R) =A*C _(Alb) +B

For blank sample (normal urine; 0 mg/L albumin):

A _(S) ^(o) −A _(R) ^(o) =A*C _(Alb) +B  (2)

For spiked sample:

A _(S) ^(*) −A _(R) ^(*) =A*C _(Alb) ^(*) +B  (3)

If the result of the spiked sample is normalized with the blank sample(i.e. equation (3) minus equation (2)), equation (4) is obtained.

(A _(S) ^(*) −A _(S) ^(o))−(A _(R) ^(*) −A _(R) ^(o))=A*(C _(Alb) ^(*)−C _(Alb) ^(o))  (4)

Assuming that A_(R) ^(*)=A_(R) ^(o), i.e. the absorbance of thereferencing reagent is the same for the Blank and Spiked samples, thenequation (4) can be rewritten as equation (5):

A _(S) ^(*) −A _(S) ^(o) =A*(C _(Alb) ^(*) −C _(Alb) ^(o)  (5)

While it is possible to measure A_(S) ^(*) and A_(R) ^(*), A_(S) ^(o) isan unknown. However, a strong correlation is found between A_(S) ^(o)and A_(R) ^(o), which counters the urine matrix effect in different realurine samples as shown in FIG. 13 and equation (6).

A _(S) ⁰ =a*A _(R) ^(o) +b=a*A _(R) ^(*) +b  (6)

By substituting equation (6) into equation (5), a new calibration curvefor the albumin assay can be obtained using equation (7) (whereinC_(Alb) ^(*) is taken as being 0 mg/L).

A _(S) ^(*)−(a*A _(R) ^(*) +b)=A*C _(Alb)  (7)

The normalised curve calibration curve of FIG. 14 is more accurateacross the concentration range, and especially at low concentrations ofalbumin. This is illustrated in Table 6.

TABLE 6 Quantified [Albumin] Spiked Vol. # Un-spiked 100 mg/L 1 −1 89 29 110 3 1 100 4 1 100 5 −6 93 6 0 101 7 −2 95 8 2 94 9 2 102 10  −8 96Average 0 98 Stdev 4.69 5.81

The above-described calibration process may be implemented as one ormore software modules executed by a standard computer system such as anIntel IA-32 based personal computer system, as shown in FIG. 16.However, it will be apparent to those skilled in the art that at leastparts of the calibration process could alternatively be implemented inpart or entirely in the form of one or more dedicated hardwarecomponents, such as application-specific integrated circuits (ASICs),and/or field programmable gate arrays (FPGAs), for example.

As shown in FIG. 16, a system 1600 for generating a calibration curvefor use in quantitatively determining the concentration of albumin in aurine sample executes a calibration process, as described above, whichis implemented as one or more software components 1630 stored onnon-volatile (e.g., hard disk, solid-state drive, or flash memory)storage 1602 associated with a standard computer system. The system 1600includes standard computer components, including random access memory(RAM) 1604, at least one processor 1606, and external interfaces 1608,1610, 1612, all interconnected by a bus 1614.

The external interfaces include universal serial bus (USB) interfaces1608, at least one of which is connected to a keyboard 1616 and apointing device such as a mouse 1618, a network interface connector(NIC) 1610 which can be used to connect the system 1600 to acommunications network such as the Internet, and a display adapter 1612,which is connected to a display device such as an LCD panel display1620. The system 100 also includes a number of standard softwarecomponents, including an operating system 1628 such as Linux orMicrosoft Windows, and a statistical software package 1629 such asMatlab or R.

The system 1600 stores, on non-volatile storage 1602, absorbance data1640, which comprise first absorbance data representing the albumin-freesample and reference absorbances A_(S) ^(o) and A_(R) ^(o), and secondabsorbance data representing the spike-in sample and referenceabsorbances A_(S) ^(*) and A_(R) ^(*). The absorbance data 1640 may bestored from previous experiments, or may be obtained in real time fromabsorbance measurements carried out on albumin-free and spike-in samplesusing optical modules 700 and communicated to system 1600 via NIC 1610,for example.

The software components 1630 may comprise a first fitting componentconfigured to computationally fit, using statistical software package1629 for example, the functional relationship described in Eq. (6)between the albumin-free sample absorbances and the albumin-freereference absorbances to obtain adjustment parameters a and b. Anabsorbance-adjusting component of components 1630 may be configured toadjust the reference absorbances using the adjustment parameters, tothereby obtain adjusted reference absorbances A′_(R)=a*A_(R) ^(*)+b; anda second fitting component of components 1630 may be configured tocomputationally fit, again using statistical software package 1629, thefunctional relationship described by Eq. (7) between the sampleabsorbances, the adjusted reference absorbances and the known (spike-in)concentrations to obtain the parameters a, b and A of the calibrationcurve. Accordingly, when it is desired to calculate an albuminconcentration C_(Alb) for a sample under test, given measuredabsorbances A_(S) and A_(R) for the sample, C_(Alb) can be calculatedaccording to A_(S)−(a*A_(R)+b)=A*C_(Alb) using the calibrationparameters a, b and A obtained by the calibration software components1630 of system 1600.

FIG. 15 shows the calibration curve for the creatinine assay. Thekinetics of the DNBA/creatinine reaction is measured and a calibrationcurve for creatinine is generated by plotting the rate of reaction vs.creatinine concentration. Creatinine standards are prepared withartificial or pooled urine and certain concentration of creatinine.

The creatinine assay requires 5 min to 10 min and microalbuminuria assayrequires around 5 min.

For the creatinine assay, it will be appreciated that a kineticmeasurement may start from the time that the sample is added to thecreatinine assay formulation, or that the measurement may be delayed(e.g. for 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes or 5minutes) before the kinetic measurements are started. Therefore, theactual window of sensing time where the measurements are taken may befrom 1 minute to 10 minutes. The time interval for data acquisitionduring the kinetic measurements in the creatinine assay may be from 0.1seconds to 15 seconds (e.g. 5 seconds).

Total assay time from sampling to provide ACR value can be less than 10min, and this is a rapid measurement.

Advantages of Dry Reagent Assay System

The dry-reagents are very easy to be reconstituted by addition of aurine sample.

The reagents allow for a one-step sensing protocol, which significantlyimproves user-friendliness.

As mentioned hereinbefore, the formulations prevent the formation ofair-bubbles adhered to the surface of the cuvette wall upon thereconstitution of the dry-reagent with a urine specimen, allowing directoptical measurement. That is, the formulation disclosed herein allowsfor fast optical measurement, without having to remove bubbles from thesolution (if even possible), the removal of bubbles from thereconstituted formulation in urine also allows for greater accuracy ofmeasuring absorbance, as it reduced light scattering.

When supplied as a dry-reagent, the formulations discussed herein areeasy to store and transport, as compared with the wet-reagents.

Operative Variations and Alternatives

The present urinalysis system described above, applying a bufferaddition method and/or a protein denaturing method can also be appliedfor urinary components other than creatinine or albumin. Examples ofsuch analytes include, but is not limited to, water, ions (e.g., sodium,potassium, chloride, phosphoric acid, phosphorus, sulfur, bromide,fluoride, iodide, calcium, magnesium, iron, lead, mercury, etc.),proteins (e.g. haptoglobin, transferrin, immunoglobulin,lactadehydrogenase, gamma-glutamyl transferase, alpha amylase,uropepsinogene, lysozyme, urokinase, etc.), sugars (e.g., glucose,phenylpyruvate, arabinose, xyloseribose, fucose, rhammose, ketopentose,galactose, mannose, fructose, lactose, sucrose, fucosylglucose,raffinose, etc.), amino acids (e.g., alanine, carnosine, glycine,histidine, leucine, lysine, methionine, phenylalanine, serine, tyrosine,valine, hydroxyloproline, galactosylhydroxylyzine, xylosylserine, etc.),hormones (e.g., epinephrine, norepinephrine, dopamine, serotonin, hCG,EFT, estradiol, gonadotropin, corticotropin, prolactin, oxytocin,vasopressin, thyroxine, catecholamines (epinephrine, norepinephrine,dopamine), insulin, erythropoietin, corticosteroids (aldosterone,corticosterone, cortisone), testosterone, progesterone, estrogen, etc.),vitamins (e.g., vitamin B1, vitamin B2, vitamin B6, 4-pyridoxique acid,nicotinic acid, vitamin B]2, biopterine, vitamin C, etc.), drugs orother bioactive substances (e.g., caffeine, cocaine, etc.), metabolicwastes (e.g., urea, uric acid, creatine, choline, piperidine, bilirubin,allantoin, etc.), ketones, folic acid, and the like.

Urinalysis can be performed for human urine with a ranging pH from 5 to8. Most people have a urine pH that is within this range, but some maybe out of the range.

The sensing temperature can be from 15° C. to 40° C.

The dry reagent can be packed within a moisture-proof bag and stored atroom temperature for a year at least.

The dry reagent system includes a reagent for reference sample to lessenthe effect of pH, temperature and interferences. For creatinine, albuminor other target assay, to include chemical with buffering effect intoreagent for reference sample is effective. Especially for albumin assay,denaturing albumin by surfactant in reference sample is highly effectiveto measure reference sample.

Wet reagents may be used instead of dry reagents. In this case, the wetreagent for measuring creatinine doesn't require a bulking agent, andthe wet reagent for measuring microalbuminuria doesn't require non-ionicsurfactant 2 as shown in Tables 7 and 8 below.

TABLE 7 Range of Concentration Chemical Category Creatinine-Samplingreagent Strong base  40-80 g/L Buffer 50-250 g/L  Anionic surfactant0.1-10 g/L Cationic surfactant 0.1-10 g/L DNBA   1-5 g/L

TABLE 8 Range of Concentration Albumin- Albumin- Chemical CategorySampling reagent Referencing reagent BCG 0.1-1.5 g/L 0.1-1.5 g/L Strongbase 10-50 g/L 10-50 g/L Buffer 50-250 g/L 50-250 g/L Non-ionicsurfactant 1 1-20 g/L 1-20 g/L Preservative 0.1-3 g/L 0.1-3 g/L Anionicsurfactant 0 w % 0.1-5 w % (1-50 g/L)

In addition, the microalbuminuria/creatinine assay can be separated.That is, the microalbuminuria assay may be run alone or the creatinineassay may also be run alone.

1-64. (canceled)
 65. A method of quantitatively determining the concentration of at least one first analyte in a sample, the method comprising the steps of: (a) adding a portion of the sample to a first analyte assay formulation comprising a first analyte complexing reagent to generate a first analyte sample; (b) adding a portion of the sample to an analyte assay reference formulation that is identical to the first analyte assay formulation, except that it further comprises a reagent that denatures the analyte or blocks the formation of a complex between the analyte and the first analyte complexing reagent, to generate an analyte reference sample; and (c) determining the concentration of the at least one first analyte in the sample by measuring the absorbance spectra of the first analyte sample and analyte reference sample, calculating the difference between the absorbance spectra of the first analyte sample and the analyte reference sample and comparing the difference spectrum obtained to a first pre-determined calibration curve of analyte concentration; wherein the at least one first analyte is albumin.
 66. The method of claim 65, wherein the method further comprises determining the concentration of at least one second analyte in the sample, the method comprising the steps of: (a) adding a portion of the sample to a second analyte assay formulation comprising a second analyte complexing reagent to generate a second analyte sample; and (b) determining the concentration of the at least one second analyte in the sample by measuring the absorbance spectra of the analyte in the second analyte sample over a period of time, calculating the rate of the absorbance change of the second analyte sample over said period of time and comparing the rate obtained to a second pre-determined calibration curve of analyte concentration.
 67. The method of claim 65, wherein the at least one first analyte is albumin and the method comprises the steps of: adding a portion of the sample to each of an: A) albumin assay sample formulation comprising an albumin complexing reagent to generate an albumin sample; and B) albumin assay reference formulation that is identical to the albumin assay formulation except that it further comprises an anionic surfactant to generate an albumin reference sample, and determining the concentration of albumin in the sample by measuring the absorbance spectra of the albumin sample and the albumin reference sample, calculating the difference between the absorbance spectra of the albumin sample and the albumin reference sample and comparing the difference spectrum obtained to a pre-determined calibration curve of albumin concentration.
 68. The method of claim 66, wherein the at least one second analyte is creatinine and the method comprises the steps of adding a portion of the sample to a creatinine assay sample formulation comprising a creatinine complexing reagent to generate a creatinine sample and determining the concentration of creatinine in the sample by measuring the change of the absorbance spectra of the creatinine sample over a period of time, calculating the rate of the absorbance change of the creatinine sample over said period of time and comparing the rate obtained to a pre-determined calibration curve of creatinine concentration.
 69. The method of claim 66, wherein the at least one first analyte is albumin and the at least one second analyte is creatinine, the method comprising the steps of: a) determining the concentration of creatinine in the sample in accordance with the method of claim 66; and b) determining the concentration of albumin in the sample in accordance with the method of: adding a portion of the sample to a first analyte assay formulation comprising a first analyte complexing reagent to generate a first analyte sample; adding a portion of the sample to an analyte assay reference formulation that is identical to the first analyte assay formulation, except that it further comprises a reagent that denatures the analyte or blocks the formation of a complex between the analyte and the first analyte complexing reagent, to generate an analyte reference sample; and determining the concentration of the at least one first analyte in the sample by measuring the absorbance spectra of the first analyte sample and analyte reference sample, calculating the difference between the absorbance spectra of the first analyte sample and the analyte reference sample and comparing the difference spectrum obtained to a first pre-determined calibration curve of analyte concentration; wherein the at least one first analyte is albumin; and further comprising the step of determining the albumin/creatinine ratio of the sample.
 70. The method of claim 65, wherein one or more of the formulations are lyophilised.
 71. The method of claim 67, wherein the albumin assay formulation is an aqueous solution that comprises: a compound that reacts with albumin to generate an albumin complex in a concentration of from about 0.1 to about 1.5 g/L; a strong base in a concentration of from about 10 to about 50 g/L; a buffer in a concentration of from about 50 to about 250 g/L; a first non-ionic surfactant in a concentration of from about 1 to about 20 g/L; and a preservative in a concentration of from about 0.1 to about 3 g/L.
 72. The method of claim 67, wherein the albumin assay reference formulation is identical to the albumin assay sample formulation comprising a compound that reacts with albumin to generate an albumin complex in a concentration of from about 0.1 to about 1.5 g/L; a strong base in a concentration of from about 10 to about 50 g/L; a buffer in a concentration of from about 50 to about 250 g/L; a first non-ionic surfactant in a concentration of from about 1 to about 20 g/L; and a preservative in a concentration of from about 0.1 to about 3 g/L, but further comprises an anionic surfactant.
 73. The method of claim 71, wherein the compound that reacts with albumin to generate an albumin complex is bromocresol green.
 74. The method of claim 71, wherein the albumin assay sample formulation and the albumin assay reference formulation are lyophilised.
 75. The method of claim 74, wherein the albumin assay sample formulation and the albumin assay reference formulation further comprise a second non-ionic surfactant in a concentration of from about 10 to about 200 g/L.
 76. The method of claim 65, wherein the pre-determined calibration curve of albumin concentration is obtained by a computer-implemented method of generating a calibration curve for use in quantitatively determining the concentration of albumin in a urine sample, the method comprising the steps of: obtaining first absorbance data representing albumin-free sample absorbances and albumin-free reference absorbances for a plurality of albumin-free urine samples; wherein the albumin-free sample absorbances comprise absorbances for respective first portions of the albumin-free urine samples in the presence of an albumin-complexing reagent; and the albumin-free reference absorbances comprise absorbances for respective second portions of the albumin-free urine samples in the presence of the albumin-complexing reagent, and additionally in the presence of an albumin-denaturing reagent or a reagent that blocks the formation of a complex between albumin and the albumin-complexing reagent; computationally fitting a functional relationship between the albumin-free sample absorbances and the albumin-free reference absorbances to obtain adjustment parameters; obtaining second absorbance data representing sample absorbances and reference absorbances for a plurality of urine samples having known concentrations of albumin; wherein the sample absorbances comprise absorbances for respective first portions of the urine samples in the presence of the albumin-complexing reagent; and the reference absorbances comprise absorbances for respective second portions of the urine samples in the presence of the albumin-complexing reagent, and additionally in the presence of the albumin-denaturing reagent or the reagent that blocks the formation of a complex between albumin and the albumin-complexing reagent; adjusting the reference absorbances using the adjustment parameters, to thereby obtain adjusted reference absorbances; and computationally fitting a functional relationship between the sample absorbances, the adjusted reference absorbances and the known concentrations to obtain parameters of the calibration curve.
 77. The method according to claim 76, wherein the functional relationship between the albumin-free sample absorbances and the albumin-free reference absorbances is of the form A_(S) ⁰=a*A_(R) ⁰+b, where A_(S) ⁰ are the albumin-free sample absorbances, A_(R) ⁰ are the albumin-free reference absorbances, and a and b are the adjustment parameters.
 78. The method according to claim 77, wherein the adjusted reference absorbances are calculated according to a*A_(R) ^(*)+b, where A_(R) ^(*) are the reference absorbances.
 79. The method according to claim 78, wherein the functional relationship between the sample absorbances, the adjusted reference absorbances and the known concentrations is of the form A_(S) ^(*)−(a*A_(R) ^(*)+b)=A*C_(Alb) ^(*), where A_(S) ^(*) are the sample absorbances, and C_(Alb) ^(*)are the known concentrations, such that a, b and A are the parameters of the calibration curve.
 80. The method of claim 68, wherein the creatinine sample assay formulation is an aqueous solution that comprises: a compound that reacts with creatinine to generate a creatinine complex a strong base in a concentration of from about 40 to about 80 g/L; a buffer in a concentration of from about 50 to about 250 g/L; and at least one surfactant in a concentration of from about 0.1 to about 20 g/L.
 81. The method of claim 80, wherein the compound that reacts with creatinine to generate a creatinine complex is dinitrobenzoic acid or picric acid.
 82. The method of claim 81, wherein the at least one surfactant of the creatinine assay sample formulation further comprises an anionic surfactant in a concentration from about 0.1 to about 10 g/L and a cationic surfactant in a concentration from about 0.1 to about 10 g/L, optionally wherein the ratio of cationic surfactant:anionic surfactant is 1:5.
 83. The method claim 80, wherein the creatinine assay sample formulation is lyophilised and, optionally, wherein the creatinine assay sample formulation further comprises a bulking agent in an amount of from about 1 to about 40 weight % of the formulation.
 84. A formulation for use in the analysis of the creatinine concentration of a sample comprising: a strong base in a concentration of from about 40 to about 80 g/L; a buffer in a concentration of from about 50 to about 250 g/L; at least one surfactant in a concentration of from about 0.1 to about 20 g/L; and water; wherein the at least one surfactant comprises an anionic surfactant in a concentration from about 0.1 to about 10 g/L and a cationic surfactant in a concentration from about 0.1 to about 10 g/L, optionally wherein the ratio of cationic surfactant: anionic surfactant is 1:2 to 1:10, such as from 1:3 to 1:7, or more particularly, 1:5, and the cationic surfactant includes a quaternary ammonium moiety.
 85. The formulation of claim 84, wherein the formulation further comprises a compound that reacts with creatinine to generate a creatinine complex.
 86. The formulation of claim 85, wherein the compound that reacts with creatinine to generate a creatinine complex is dinitrobenzoic acid or picric acid.
 87. The formulation of claim 84, wherein the formulation is lyophilised.
 88. The formulation of claim 87, wherein the formulation further comprises a bulking agent in an amount of from about 1 to about 40 weight % of the formulation, optionally wherein the bulking agent is selected from one or more of the group consisting of sugar-mannitol, lactose, and trehalose.
 89. A microalbuminuria formulation for use in the analysis of albumin concentration of a urine sample comprising: a compound that reacts with albumin to generate an albumin complex in a concentration of from about 0.1 to about 1.5 g/L; a strong base in a concentration of from about 10 to about 50 g/L; a buffer in a concentration of from about 50 to about 250 g/L; a first non-ionic surfactant in a concentration of from about 1 to about 20 g/L; a preservative in a concentration of from about 0.1 to about 3 g/L; and water.
 90. The formulation of claim 89, wherein the formulation further comprises an anionic surfactant.
 91. The formulation of claim 89 wherein the compound that reacts with albumin to generate an albumin complex is bromocresol green.
 92. The formulation of claim 89, wherein the formulation is lyophilised.
 93. The formulation of claim 90, wherein the formulation further comprises a second non-ionic surfactant in an amount of from about 10 to about 200 g/L of the formulation.
 94. A method of preparing a lyophilised formulation for use in the analysis of the creatinine concentration of a sample using the creatinine assay formulation of claim 84, the method comprising the steps of: (a) mixing the prescribed chemical components of the formulation together, filtering the resultant mixture, dispensing said mixture into a container and inserting the container into a freeze drying apparatus; (b) freezing the formulation at a temperature of from about −20° C. to about −80° C. for a period of time ranging from about 0.5 hours to 5 hours; (c) annealing the formulation at a temperature of from about −10° C. to about −30° C. for a period of time ranging from about 1 hour to 5 hours; (d) re-freezing the formulation at a temperature of from about −20° C. to about −80° C. for a period of time ranging from about 0.5 hours to 5 hours; (e) conducting a first drying cycle at a temperature of from about −10° C. to about −30° C. for a period of time ranging from about 5 hours to 50 hours; and (f) conducting a second drying cycle at a temperature of from about 0° C. to about 60° C. for a period of time ranging from about 1 hour to 20 hours.
 95. The method of claim 94, wherein the method further comprises storing the container holding the lyophilised formulation away from light, oxygen and moisture.
 96. A kit of parts comprising one or both of: (a) a creatinine assay formulation comprising: a strong base in a concentration of from about 40 to about 80 g/L; a buffer in a concentration of from about 50 to about 250 g/L; at least one surfactant in a concentration of from about 0.1 to about 20 g/L; and water; wherein the at least one surfactant comprises an anionic surfactant in a concentration from about 0.1 to about 10 g/L and a cationic surfactant in a concentration from about 0.1 to about 10 g/L, optionally wherein the ratio of cationic surfactant: anionic surfactant is 1:2 to 1:10, such as from 1:3 to 1:7, or more particularly, 1:5, and the cationic surfactant includes a quaternary ammonium moiety; and (b) an albumin assay formulation comprising: a compound that reacts with albumin to generate an albumin complex in a concentration of from about 0.1 to about 1.5 g/L; a strong base in a concentration of from about 10 to about 50 g/L; a buffer in a concentration of from about 50 to about 250 g/L; a first non-ionic surfactant in a concentration of from about 1 to about 20 g/L; a preservative in a concentration of from about 0.1 to about 3 g/L; and water; and an albumin reference formulation according to the formulation of claim
 90. 97. The kit of parts of claim 96, wherein the formulations are stored away from light, oxygen and moisture.
 98. A computer-implemented method of generating a calibration curve for use in quantitatively determining the concentration of albumin in a urine sample, the method comprising the steps of: obtaining first absorbance data representing albumin-free sample absorbances and albumin-free reference absorbances for a plurality of albumin-free urine samples; wherein the albumin-free sample absorbances comprise absorbances for respective first portions of the albumin-free urine samples in the presence of an albumin-complexing reagent; and the albumin-free reference absorbances comprise absorbances for respective second portions of the albumin-free urine samples in the presence of the albumin-complexing reagent, and additionally in the presence of an albumin-denaturing reagent or a reagent that blocks the formation of a complex between albumin and the albumin-complexing reagent; computationally fitting a functional relationship between the albumin-free sample absorbances and the albumin-free reference absorbances to obtain adjustment parameters; obtaining second absorbance data representing sample absorbances and reference absorbances for a plurality of urine samples having known concentrations of albumin; wherein the sample absorbances comprise absorbances for respective first portions of the urine samples in the presence of the albumin-complexing reagent; and the reference absorbances comprise absorbances for respective second portions of the urine samples in the presence of the albumin-complexing reagent, and additionally in the presence of the albumin-denaturing reagent or the reagent that blocks the formation of a complex between albumin and the albumin-complexing reagent; adjusting the reference absorbances using the adjustment parameters, to thereby obtain adjusted reference absorbances; and computationally fitting a functional relationship between the sample absorbances, the adjusted reference absorbances and the known concentrations to obtain parameters of the calibration curve.
 99. A system for generating a calibration curve for use in quantitatively determining the concentration of albumin in a urine sample, the system comprising: a memory for storing first absorbance data representing albumin-free sample absorbances and albumin-free reference absorbances for a plurality of albumin-free urine samples; wherein the albumin-free sample absorbances comprise absorbances for respective first portions of the albumin-free urine samples in the presence of an albumin-complexing reagent; and the albumin-free reference absorbances comprise absorbances for respective second portions of the albumin-free urine samples in the presence of the albumin-complexing reagent, and additionally in the presence of an albumin-denaturing reagent or a reagent that blocks the formation of a complex between albumin and the albumin-complexing reagent; said memory further storing second absorbance data representing sample absorbances and reference absorbances for a plurality of urine samples having known concentrations of albumin; wherein the sample absorbances comprise absorbances for respective first portions of the urine samples in the presence of the albumin-complexing reagent; and the reference absorbances comprise absorbances for respective second portions of the urine samples in the presence of the albumin-complexing reagent, and additionally in the presence of the albumin-denaturing reagent or the reagent that blocks the formation of a complex between albumin and the albumin-complexing reagent; a first fitting component configured to computationally fit a functional relationship between the albumin-free sample absorbances and the albumin-free reference absorbances to obtain adjustment parameters; an absorbance-adjusting component configured to adjust the reference absorbances using the adjustment parameters, to thereby obtain adjusted reference absorbances; and a second fitting component configured to computationally fit a functional relationship between the sample absorbances, the adjusted reference absorbances and the known concentrations to obtain parameters of the calibration curve.
 100. The method of claim 66, wherein one or more of the formulations are lyophilised.
 101. The method of claim 67, wherein one or more of the formulations are lyophilised.
 102. The method of claim 68, wherein one or more of the formulations are lyophilised.
 103. The method of claim 69, wherein one or more of the formulations are lyophilised.
 104. A method of preparing a lyophilised formulation for use in the analysis of albumin concentration of a sample using the formulation of claim 89, the method comprising the steps of: (a) mixing the prescribed chemical components of the formulation together, filtering the resultant mixture, dispensing said mixture into a container and inserting the container into a freeze drying apparatus; (b) freezing the formulation at a temperature of from about −20° C. to about −80° C. for a period of time ranging from about 0.5 hours to 5 hours; (c) annealing the formulation at a temperature of from about −10° C. to about −30° C. for a period of time ranging from about 1 hour to 5 hours; (d) re-freezing the formulation at a temperature of from about −20° C. to about −80° C. for a period of time ranging from about 0.5 hours to 5 hours; (e) conducting a first drying cycle at a temperature of from about −10° C. to about −30° C. for a period of time ranging from about 5 hours to 50 hours; and (f) conducting a second drying cycle at a temperature of from about 0° C. to about 60° C. for a period of time ranging from about 1 hour to 20 hours.
 105. The method of claim 104, wherein the method further comprises storing the container holding the lyophilised formulation away from light, oxygen and moisture. 